Nitric oxide (NO) generated by endothelial NO synthase (eNOS) is a fundamental signaling moleculeand effector in cardiovascular regulation and disease. NO synthesis from eNOS is critically regulated by several scaffolding proteins. Notably, caveolin-1, the main protein component of caveolae in endothelial cells, inhibits eNOS activity. Conversely, molecular chaperon heat shock protein 90 (hsp90) enhances eNOS function. Phosphorylation of eNOS, such as at serine 1179 and threonine 497 of bovine eNOS, profoundly influences NO synthesis. Besides NO, the applicant and others recently discovered that eNOS also produces superoxide (.O2-), a primary reactive oxygen species. There is increasing evidence showing that .O2- generation from eNOS plays important roles in the pathogenesis of endothelial dysfunction, a common cause of various cardiovascular diseases. However, exactly what mechanisms cause eNOS to produce .O2-is largely unknown. To reveal the roles of eNOS-derived .O2- in cardiovascular regulation and disease, the modulating mechanisms of .O2- generation from this enzyme must be understood first. The applicant's preliminary studies showed that .O2- generation from eNOS was strongly influenced by caveolin-1, hsp90, and phosphorylation. Therefore, it is hypothesized that .O2- generation from eNOS is critically modulated by protein-protein interactions and protein phosphorylation. To examine this hypothesis, the following specific aims are proposed. 1) To determine the effects of caveolin-1 and hsp90 on .O2- generation from purified eNOS. 2) To study the effects of protein phosphorylation on .O2- generation from eNOS. 3) To define the roles of caveolin-1, hsp90, and protein phosphorylation in modulating .O2- generation from eNOS in cells. 4) To determine the effects of caveolin-1, hsp90, and protein phosphorylation on .O2- generation from eNOS in aortas of rats and transgenic mice. For each of these aims, electron paramagnetic resonance measurements of .O2- will be combined with molecular, cellular, and physiological approaches to characterize the regulation of .O2- generation from eNOS in vitro and in vivo. Results from these studies will provide fundamental mechanistic information regarding how .O2- generation from eNOS is regulated. This information may lead to new approaches to treat endothelial dysfunction and prevent cardiovascular diseases.